Regulation of motor proteins, axonal transport deficits and adult-onset neurodegenerative diseases

ABSTRACTFollowing its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21’s contribution to PRV neuroinvasionin vivo. Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transportin vitroandin vivo. Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.IMPORTANCEHerpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasionin vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

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Glutamate Slows Axonal Transport of Neurofilaments in Transfected Neurons

The Journal of Cell Biology ◽

10.1083/jcb.150.1.165 ◽

2000 ◽

Vol 150 (1) ◽

pp. 165-176 ◽

Cited By ~ 108

Author(s):

Steven Ackerley ◽

Andrew J. Grierson ◽

Janet Brownlees ◽

Paul Thornhill ◽

Brian H. Anderton ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Axonal Transport ◽

Fluorescent Protein ◽

Mitogen Activated Protein Kinase ◽

Glutamate Excitotoxicity ◽

Slow Axonal Transport ◽

Mitogen Activated Protein ◽

Green Fluorescent ◽

Pathogenic Process ◽

Neurofilament Transport

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein–tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.

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Effects of the dynein inhibitor dynarrestin on the number of motor proteins transporting synaptic cargos

10.1101/2020.02.07.934687 ◽

2020 ◽

Author(s):

Kumiko Hayashi ◽

Miki G. Miyamoto ◽

Shinsuke Niwa

Keyword(s):

Axonal Transport ◽

Optical Tweezers ◽

Hippocampal Neurons ◽

Motor Proteins ◽

Force Measurement ◽

Retrograde Transport ◽

Long Distance ◽

Anterograde Transport ◽

Non Invasive ◽

Physical Measurements

AbstractSynaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated using non-invasive measurements of transport force based on non-equilibrium statistical mechanics. Although direct physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the non-invasive estimations enabled enumerating force producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than anterograde transport. In the future, these measurements may be used to quantify the damage to axonal transport resulting from neuronal diseases including Alzheimer’s, Parkinson’s, and Huntington’s diseases.

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